Inhibition of heparin-binding

ABSTRACT

The invention provides a method for extending the plasma half-life of heparin-binding proteins by coadministering such proteins with a therapeutically acceptable compound capable of inhibiting their binding to a low affinity heparin-like binding site on the surface of cells. In one embodiment of the invention, the-heparin-binding protein is a selectin. The binding inhibitory compound can, for example, be a purified native heparin preparation, a heparin fragment, or another polyanionic compound, such as dextran sulfate, heparan sulfate, pentosan sulfate, or hyaluronate.

The present application is a continuation-in-part of application Ser.No. 08/118,162, filed 8 Sep. 1993, now abandoned.

BACKGROUND OF THE INVENTION

1.Field of the Invention

The present invention concerns improvements in the purification, testingand therapeutic use of heparin-binding proteins, based upon theinhibition of the interaction between such proteins and naturallyoccurring heparin-like substances. More specifically, the inventionrelates to improved methods and compositions for the administration ofheparin-binding proteins. The invention further relates to improvedmethods for the purification and for testing the biological activity ofheparin-binding proteins.

2. Description of Background and Related Art

It is known that a large variety of naturally occurring, biologicallyactive polypeptides bind heparin. Such heparin-binding polypeptidesinclude cytokines (also termed chemokines), such as platelet factor 4and IL-8 (Barber et al., Biochim. Biophys. Acta 286, 312-329 (1972);Handin et al., J. Biol. Chem. 251, 4273-422 (1976); Loscalzo et al.,Arch. Biochem. Biophys. 240, 446-455 (1985); Zucker et al., Proc. Natl.Acad. Sci. USA 86, 7571-7574 (1989); Talpas et al., Biochim. Biophys.Acta 1078, 208-218 (1991); Webb et al., Proc. Natl. Acad. Sci. USA 90,7158-7162 (1993)); heparin-binding growth factors (Burgess and Maciag,Annu. Rev. Biochem. 58, 576-606 (1989); Klagsbrun, Prog. Growth FactorRes. 1, 207-235 (1989)) , such as epidermal growth factor (EGF);platelet--derived growth factor (PDGF); basic fibroblast growth factor(bFGF); acidic fibroblast growth factor (aFGF); vascular endothelialgrowth factor (VEGF); and hepatocyte growth factor (HGF) (for the lattersee also Liu et al., Am. J. Physiol. 263 (Gastrointest. Liver Physiol.26): G642-G649 (1992)); and selectins, such as L-selectin, E-selectinand P-selectin (Norgard-Sumnicht et al., Science 261, 480-483 (1993)) .

The preparation of homogeneously sized, heparin-derived oligosaccharidesprepared from heparin has been reported, and heparin-derived hexa- andoctasaccharides were described to be able to inhibit the interactionbetween cell surface heparan proteoglycan and bFGF (Ishihara et al., J.Biol. Chem. 268, 4675-4683 (1993)). The structure of a specificheparin-derived hexasaccharide showing high affinity for bFGF wasdescribed by Tyrrell et al., J. Biol. Chem. 268, 4684-4689 (1993).

The heparin-binding proteins are typically cleared rapidly from theplasma in vivo, and this rapid clearance greatly limits theirtherapeutic applications. Although the liver and the kidney have beenidentified as the major clearance organs for a number of heparin-bindingproteins (see, e.g. Kim et al., J. Pharm. Sci. 77, 200-207 (1988);Sugiyama et al., Pharm. Res. 6, 194-204 (1989), Sugiyama et al., J.Controlled Release 13, 157-174 (1990) and Sugiyama et al., ReceptorMediated Hepatic Clearance of Peptide Hormones, In: Topics inPharmaceutical Sciences 1989, Breimer et al., eds., Elsevier, New York,1989, p. 429-443), little is known about the mechanism of their rapidremoval from the circulation. In a recent publication (Liu et al.,supra) it was proposed that there are at least two different kinds ofbinding sites for HGF on the surface of hepatic cells: aheparin-washable binding site with lower affinity and aheparin-resistant, acid-washable binding site with higher affinity, andthat the low-affinity binding site appears to play a role in theinternalization of HGF. The binding of other heparin-binding proteins totheir respective cell surface receptors was described to be dependentupon heparin-like molecules (see, e.g. Gitay-Goren et al., J. Biol.Chem. 267, 6093-6098 (1992) for VEGF), and it was shown that theaddition of exogenous heparin is able to potentiate the binding ofcertain heparin-binding proteins to their receptors.

It would be desirable to increase the plasma half-life and/or decreasethe plasma clearance of heparin-binding proteins.

It would further be desirable to improve the therapeutic potency ofheparin-binding proteins.

It would also be desirable to improve the bioavailability of suchpolypeptides when they are not directly administered into the bloodstream.

It would additionally be desirable to prevent the inactivation ofheparin-binding polypeptides by other binding proteins in blood.

It would further be desirable to enhance the sensitivity of assays forthe detection of heparin-binding proteins.

It would be also desirable to enhance the purification ofheparin-binding proteins from cell cultures.

Accordingly, it is an object of the present invention to provide amethod for extending the plasma half-life of heparin-binding proteins.

It is another object of the invention to improve the therapeutic potencyof heparin-binding proteins.

It is a further object, to improve the bioavailability ofheparin-binding proteins.

It is a still further object to provide methods for preventing theinactivation of heparin-binding proteins by other blood proteins.

It is yet another object to provide new and improved pharmaceuticalcompositions of heparin-binding proteins.

It is an additional object to enhance the sensitivity of assays for thedetection of heparin-binding proteins.

It is a still further object to facilitate the purification ofheparin-binding proteins.

It is another object of the present invention to provide methods for theintraperitoneal or subcutaneous administration of heparin-bindingproteins.

SUMMARY OF THE INVENTION

The present invention is based on the finding that the in vivo half-lifeof heparin-binding growth factors, such as HGF, in the plasma can besignificantly extended by coadministration with a polyanionic molecule,such as heparin or heparin-derived oligosaccharides. It has further beenfound that the coadministration of polyanionic molecules increases theamount of heparin-binding proteins entering the plasma afterintraperitoneal or subcutaneous administration. Although it is believedthat the effect of polyanionic molecules on the bioavailability ofheparin-binding proteins is due to their ability to block the binding ofheparin-binding proteins to extracellular matrix glycosylaminoglycans,the invention is not limited by this or any other theory in any way. Theinvention is additionally based on the finding that the presence ofpolyanionic molecules, and specifically heparin and heparin-likeoligosaccharides potentiates the biological activity of heparin-bindingproteins (HGF, IL-8) and/or enhances their binding to their respectivenative receptors (VEGF).

In one aspect, the invention concerns a method for extending the plasmahalf-life of a heparin-binding protein comprising coadministering suchprotein with a therapeutically acceptable compound capable of inhibitingits binding to a low affinity heparin-like binding site on the surfaceof cells of an organ highly perfused with blood.

In another aspect, the invention concerns a method for improving thetherapeutic potency of a heparin-binding protein comprisingcoadministering with a therapeutically effective amount of the protein atherapeutically acceptable polyanionic compound.

In a further aspect, the invention concerns a method of improving thebioavailability of a heparin-binding protein comprising coadministeringthe protein with a therapeutically acceptable compound capable ofinhibiting its interaction with heparin-like substances in theextracellular matrix.

In a still further aspect, the invention concerns a method forpreventing the inactivation of a heparin-binding protein by a serumprotein, which comprises coadministering the heparin-binding proteinwith a polyanionic compound, such as dextran sulfate, heparin sulfate,heparan sulfate or a functional derivative thereof.

In yet another aspect, the invention concerns a pharmaceuticalcomposition comprising a therapeutically effective amount of aheparin-binding protein in association with a polyanionic compound.

In an additional aspect, the invention concerns a method for enhancingthe sensitivity of an assay for the detection of a heparin-bindingprotein in a test sample, which method comprises the addition of apolyanionic compound to the test sample.

In a further aspect, the invention concerns a method for thepurification of a heparin-binding protein from a cell culture comprisingsupplementing the cell culture with a compound capable of inhibiting thebinding of the heparin-binding protein to heparin-like substancestherein.

In a still further aspect, the invention concerns a method for theinduction of hepatocyte growth by administering to a patient in need ofsuch treatment a therapeutically effective amount of hepatocyte growthfactor (HGF) in combination with a polyanionic compound, preferably adextran or dextran sulfate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The effect of heparin on the biological activity of HGF.

FIG. 2. The effect of heparin on the detection of HGF in an ELISA.

FIG. 3. The effect of heparin on the pharmacokinetics of HGF in vivo.

FIG. 4. The effect of polyanions on the bioavailability of HGF aftersubcutaneous administration.

FIGS. 5A-5B. The effect of heparin on the hepatic clearance of [¹²⁵I]HGF.

FIG. 6. HGF/SF dose-response. Osmotic minipumps loaded to deliver theindicated doses of HGF/SF and/or dextran sulfate were implanted in theperitoneal cavity of adult mice. Three days later, plasma HGF/SF andliver weight were determined. Bars and symbols represent means ± SEM for6 animals. Bars identified by different letters are significantlydifferent (P<0.05).

FIG. 7. Saturability of HGF/SF-induced liver growth. Liver weight wasplotted as a function of plasma HGF/SF for individual mice that wereinfused intraperitoneally for 3 days with saline, dextran sulfate aloneat 4.8 mg/kg/d, or HGF/SF plus dextran sulfate at doses of 1.2, 2.4, or4.8 mg/kg/d.

FIGS. 8A-8C. Histology. Representative histological sections of liverfrom control animals (A) and from animals treated with HGF/SF plusdextran sulfate (B and C). Heavy arrows identify cells in metaphase,script arrows show cells in telophase.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

"Heparin" (also referred to a heparinic acid) is a heterogenous group ofhighly sulfated, straight-chain anionic mucopolysaccharides, calledglycosaminoglycans. Although others may be present, the main sugars inheparin are: α-L-iduronic acid 2-sulfate, 2-deoxy-2-sulfamino-α-glucose6-sulfate, β-D-glucuronic acid, 2-acetamido-2-deoxy-α-D-glucose, andL-iduronic acid. These and optionally other sugars are joined byglycosidic linkages, forming polymers of varying sizes. Due to thepresence of its covalently linked sulfate and carboxylic acid groups,heparin is strongly acidic. The molecular weight of heparin varies fromabout 6,000 to about 20,000 Da depending on the source and the method ofdetermination. Native heparin is a constituent of various tissues,especially liver and lung, and mast cells in several mammalian species.Heparin and heparin salts (heparin sodium) are commercially availableand are primarily used as anticoagulants in various clinical situations.

The term "heparin-binding protein" as used herein refers to apolypeptide capable of binding heparin (as hereinabove defined). Thedefinition includes the mature, pre, pre-pro, and pro forms of nativeheparin-binding proteins, if any, purified from natural source,chemically synthesized or recombinantly produced, their functionalderivatives and inhibitors. Typical examples of heparin-binding proteinsare growth factors, including but not limited to epidermal growth factor(EGF), platelet derived growth factor (PDGF), basic fibroblast growthfactor (bFGF), acidic fibroblast growth factor (aFGF), vascularendothelial growth factor (VEGF) , hepatocyte growth factor (HGF) (alsoknown as scatter factor, SF), NGF, IL-8, etc.

A "functional derivative" of a native heparin-binding protein is acompound that retains at least one qualitative biological activity ofthe corresponding native protein and has the ability to bind heparin.Functional derivatives include, but are not limited to, fragments ofnative heparin-binding proteins from any animal species, and derivativesof the native proteins and fragments thereof, wherein the term"derivative" is used to define amino acid sequence and glycosylationvariants, and covalent modifications of a native protein, whereas theterm "variant" refers to amino acid sequence and glycosylation variantswithin this definition. An "inhibitor" of a native heparin-bindingprotein is a compound that inhibits at least one biological activity ofthe corresponding native protein and has the ability to bind heparin.

The term "amino acid sequence variant" refers to molecules with somedifferences in their amino acid sequences as compared to a nativesequence heparin-binding protein or a fragment thereof. Ordinarily, theamino acid sequence variants will possess at least about 70%, preferablyat least about 80%, more preferably at least about 90%, most preferablyat least about 95% homology with a native heparin-binding protein, orwith a fragment thereof which, retains at least the regions required forheparin binding and for binding to the receptor(s) mediating thebiological activity of the protein. Such amino acid sequence variantsare preferably encoded by DNA capable, under stringent conditions, ofhybridizing to the complement of DNA encoding the corresponding nativeheparin-binding protein or a fragment thereof. The amino acid sequencevariants: possess substitutions, deletions, and/or insertions at certainpositions within the amino acid sequence of a native heparin-bindingprotein.

"Homology" is defined as the percentage of residues in the candidateamino acid sequence that are identical with the residues in the aminoacid sequence of a native heparin-binding protein or a fragment thereof,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent homology. Methods and computer programs forthe alignment are well known in the art.

"Stringent conditions" are overnight incubation at 37° C. in a solutioncomprising: 40% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 50° C.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative ligand sequence. Immediately adjacent to an amino acid meansconnected to either the α-carboxy or α-amino functional group of theamino acid.

Deletional variants are those with one or more amino acids in the nativeligand amino acid sequence removed. Ordinarily, deletional variants willhave one or two amino acids deleted in a particular region of themolecule.

The term "glycosylation variant" is used to refer to a heparin-bindingprotein molecule having a glycosylation profile different from that of acorresponding native protein. Glycosylation of polypeptides is typicallyeither N-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side-chain of an asparagine residue. Thetripeptide sequences, asparagine-X-serine and asparagine-X-threonine,wherein X is any amino acid except proline, are recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. O-linked glycosylation refers to the attachment of one ofthe sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyaminoacid, most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be involved in O-linked glycosylation. Anydifference in the location and/or nature of the carbohydrate moietiespresent in a glycosylation variant or fragment as compared to its nativecounterpart is within the scope herein.

The glycosylation pattern of native polypeptides can be determined bywell known techniques of analytical chemistry, including HPAEchromatography [Hardy, M. R. et al., Anal. Biochem. 170, 54-62 (1988)],methylation analysis to determine glycosyl-linkage composition[Lindberg, B., Meth. Enzymol. 28. 178-195 (1972); Waeghe, T. J. et al.,Carbohydr. Res. 123, 281-304 (1983)], NMR spectroscopy, massspectrometry, etc.

"Covalent derivatives" include modifications of a native heparin-bindingprotein or a fragment thereof with an organic proteinaceous ornon-proteinaceous derivatizing agent, and post-translationalmodifications. Covalent modifications are traditionally introduced byreacting targeted amino acid residues with an organic derivatizing agentthat is capable of reacting with selected side-chains or terminalresidues, or by harnessing mechanisms of post-translationalmodifications that function in selected recombinant host cells. Certainpost-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the heparin-binding protein moleculesas defined in the present invention. Other post-translationalmodifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl, threonyl, or tyrosylresidues, methylation of the α-amino groups of lysine, arginine, andhistidine side chains [T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86(1983)].

"Biological activity" is defined as either 1) immunologicalcross-reactivity with at least one epitope of a native heparin-bindingprotein, or 2) the possession of at least one adhesive, regulatory oreffector function qualitatively in common with a native heparin-bindingprotein.

"HGF" was identified initially as a mitogen for hepatocytes[Michalopoulos et al., Cancer Res.44, 4414-4419 (1984); Russel et al.,J. Cell. Physiol. 119, 183-192 (1984) and Nakamura et al., Biochem.Biophys. Res. Comm. 122:1450-1459 (1984)]. Nakamura et al., Suprareported the purification of HGF from the serum of partiallyhepatectomized rats. Subsequently, HGF was purified from rat platelets,and its subunit structure was determined [Nakamura et al., Proc. Natl.Acad. Sci. USA, 83, 6489-6493 (1986); and Nakamura et al., FEBS Letters224, 311-316 (1987)]. The purification of human HGF (huHGF) from humanplasma was first described by Gohda et al., J. Clin. Invest. 81, 414-419(1988).

Both rat HGF and huHGF have been molecularly cloned, including thecloning and sequencing of a naturally occurring variant lacking 5 aminoacids designated "delta5 HGF" [Miyazawa et al., Biochem. Biophys. Res.Comm. 163, 967-973 (1989); Nakamura et al., Nature 342,440-443 (1989);Seki et al, Biochem. and Biophys. Res. Commun. 172,321-327 (1990);Tashiro et al., Proc. Natl. Acad. Sci. USA 87, 3200-3204 (1990); Okajimaet al., Eur. J. Biochem. 193, 375-381 (1990)].

The mature form of huHGF, corresponding to the major form purified fromhuman serum, is a disulfide linked heterodimer derived by proteolyticcleavage of the human pro-hormone between amino acids R494 and V495.This cleavage process generates a molecule composed of an α-subunit of440 amino acids (M_(r) 69 kDa) and a β-subunit of 234 amino acids (M_(r)34 kDa). The nucleotide sequence of the hHGF cDNA reveals that both theα- and the β-chains are contained in a single open reading frame codingfor a pre-pro precursor protein. In the predicted primary structure ofmature hHGF, an interchain S-S bridge is formed between Cys 487 of theα-chain and Cys 604 in the β-chain (see Nakamura et al., Nature, supra).The N-terminus of the α-chain is preceded by 54 amino acids, startingwith a methionine group. This segment includes a characteristichydrophobic leader (signal) sequence of 31 residues and the prosequence.The α-chain starts at amino acid (aa) 55, and contains four Kringledomains. The Kringle 1 domain extends from about aa 128 to about aa 206,the Kringle 2 domain is between about aa 211 and about aa 288, theKringle 3 domain is defined as extending from about aa 303 to about aa383, and the Kringle 4 domain extends from about aa 391 to about aa 464of the α-chain. It will be understood that the definition of the variousKringle domains is based on their homology with kringle-like domains ofother proteins (prothrombin, plasminogen), therefore, the above limitsare only approximate. As yet, the function of these Kringles has notbeen determined. The β-chain of huHGF shows high homology to thecatalytic domain of serine proteases (38% homology to the plasminogenserine protease domain). However, two of the three residues which formthe catalytic triad of serine proteases are not conserved in huHGF.Therefore, despite its serine protease-like domain, hHGF appears to haveno proteolytic activity and the precise role of the β-chain remainsunknown. HGF contains four putative asparagine-linked glycosylationsites, which are located at positions 294 and 402 of the α-chain and atpositions 566 and 653 of the β-chain, in addition an O-linkedoligosaccharide is attached to Thr-445 [Shimizu et al., Biochem.Biophys. Res. Comm. 189: 1329-1335 (1992)].

In a portion of cDNA isolated from human leukocytes in-frame deletion of15 base pairs was observed. Transient expression of the cDNA sequence inCOS-1 cells revealed that the encoded HGF molecule (delta5 HGF) lacking5 amino acids in the Kringle 1 domain was fully functional (Seki et al.,supra).

A naturally occurring huHGF variant has recently been identified whichcorresponds to an alternative spliced form of the huHGF transcriptcontaining the coding sequences for the N-terminal finger and first twokringle domains of mature huHGF [Chan et al., Science 254, 1382-1385(1991); Miyazawa et al., Eur. J. Biochem. 197, 15-22 (1991)]. Thisvariant, designated HGF/NK2, has been proposed to be a competitiveantagonist of mature huHGF.

The comparison of the amino acid sequence of rat HGF with that of huHGFrevealed that the two sequences are highly conserved and have the samecharacteristic structural features. The length of the four Kringledomains in rat HGF is exactly the same as in huHGF. Furthermore, thecysteine residues are located in exactly the same positions; anindication of similar three-dimensional structures (Okajima et al.,supra; Tashiro et al., supra).

For further information about HGF see: "Hepatocyte Growth Factor--Scatter Factor (HGF-SF) and the C-Met Receptor", I. D. Goldberg and E.M. Rosen eds., Birkhauser Verlag, 1993.

As used herein, the terms "hepatocyte growth factor", "HGF" and "huHGF"refer to a (human) growth factor capable of specific binding to areceptor of wild-type (human) HGF, which growth factor typically has astructure with six domains (finger, Kringle 1, Kringle 2, Kringle 3,Kringle 4 and serine protease domains), but nonetheless may have fewerdomains or may have some of its domains repeated if it still retains itsqualitative HGF receptor binding ability. This definition specificallyincludes the delta5 huHGF as disclosed by Seki et al., supra. The terms"hepatocyte growth factor" and "HGF" also include hepatocyte growthfactor from any non-human animal species, and in particular rat HGF.

The terms "wild-type human hepatocyte growth factor", "native humanhepatocyte growth factor", "wild-type huHGF", and "native huHGF" referto native sequence human HGF, i.e., that encoded by the cDNA sequencepublished by Miyazawa, et al. 1989, supra, or Nakamura et al., 1989,supra, including its mature, pre, pre-pro, and pro forms, purified fromnatural source, chemically synthesized or recombinantly produced. Thesequences reported by Miyazawa et al.,and Nakamura et al. differ in 14amino acids. The reason for the differences is not entirely clear;polymorphism or cloning artifacts are among the possibilities. Bothsequences are specifically encompassed by the foregoing terms as definedfor the purpose of the present invention. It will be understood thatnatural allelic variations exist and can occur among individuals, asdemonstrated by one or more amino acid differences in the amino acidsequence of each individual. Amino acid positions in the variant huHGFmolecules herein are indicated in accordance with the numbering ofMiyazawa et al. 1989, supra.

The biological activity of a native HGF is shared by a functionalderivative thereof that has a qualitative mitogenic, motogenic ormorphogenic activity exhibited by a native HGF or that possesses animmune epitope that is immunologically cross-reactive with an antibodyraised against at least one epitope of the corresponding native HGF. TheHGF biological activity may, for example, be determined in an in vitroor in vivo assay of hepatocyte growth promotion. Adult rat hepatocytesin primary culture have been extensively used to search for factors thatregulate hepatocyte proliferation. Accordingly, the mitogenic effect ofan HGF variant can be conveniently determined in an assay suitable fortesting the ability of an HGF molecule to induce DNA synthesis of rathepatocytes in primary cultures, such as, for example, described inExample 2. Human hepatocytes are also available from whole liverperfusion on organs deemed unacceptable for transplantation, pare-downsof adult livers used for transplantation in children, fetal livers andliver remnants removed at surgery for other indications. Humanhepatocytes can be cultured similarly to the methods established forpreparing primary cultures of normal rat hepatocytes. Hepatocyte DNAsynthesis can, for example, be assayed by measuring incorporation of [³H]thymidine into DNA, with appropriate hydroxyurea controls forreplicative synthesis.

The effect of HGF variants on hepatocyte growth can also be tested invivo in animal models of liver dysfunction and regeneration, such as inrats following partial hepatectomy, or carbon tetrachloride causedhepatic injury, in D-galactosamine induced acute liver failure models,etc. According to a suitable protocol, a liver poison, e.g.α-naphthylisothiocyanate (ANIT) is administered to rats in apredetermined concentration capable of causing reproducible significantelevation of liver enzyme and bilirubin levels. The rats are thentreated with the HGF variant to be tested, sacrificed and the liverenzyme and bilirubin levels are determined. The livers are additionallyobserved for hepatic lesions.

As used herein, "vascular growth factor", or "VEGF" refers to amammalian growth factor derived originally from bovine pituitaryfollicular cells having the amino acid sequence disclosed in U.S. Pat.No. 5,332,671, together with functional derivatives thereof having thequalitative biological activity of a corresponding native VEGF,including, but not limited to, the human VEGF amino acid sequence. Thebiological activity of native VEGF is shared by any functionalderivative thereof that is capable of promoting selective growth ofvascular endothelial cells but not of bovine corneal endothelial cells,lens epithelial cells, adrenal cortex cells, BHK-21 fibroblasts, orkeratinocytes, or that possesses an immune epitope that isimmunologically cross-reactive with an antibody raised against at leastone epitope of the corresponding native VEGF.

The term "selectin" is used to describe cell adhesion molecules alsoreferred to as LEC-CAMs, the number of which currently stands at three:L-selectin (a.k.a. periopheral lymphone node homing receptor (pnHR),LEC-CAM-1, LAM-1, gp90^(MEL), gp100^(MEL), gp110^(MEL), MEL-14 antigen,Leu-8 antigen, TQ-1 antigen, DREG antigen); E-selectin (LEC-CAM-2,LECAM-2, ELAM-1) and P-selectin (LEC-CAM-3, LECAM-3, GMP-140, PADGEM)(see Belacqua et al., Science 243, 1160 (1989) and Geng et al., Nature343, 757 (1990)). As used herein, the term "selectin" refers to any ofthe native-sequence selectins from any (human or non-human) animalspecies, and to their functional derivatives. The biological activity ofa native selectin is shared by any functional derivative thereof thathas at least one adhesive, regulatory or effector function qualitativelyin common with a corresponding native selectin, or that showsimmunological cross-reactivity with at least one epitope of a nativeselectin.

The terms "half-life" and "plasma half-life" refer to the time by whichhalf of the administered amount of a heparin-binding protein is removedfrom the blood stream.

The terms "clearance rate" and "clearance" refer to the rate at which aheparin-binding protein is removed from the blood stream.

The expressions "biological potency" and "in vivo biological potency"are used to refer to biological activity per unit dose of aheparin-binding protein.

The term "inhibition" in connection with the binding of two substancesto one another is used in the broadest sense and includes the completeblocking of binding just as the reduction of the amount of the substancebound.

The term "therapeutically effective dose" is used to refer to an amountsufficient for the prevention or treatment of a specified physiologicalcondition or symptom. Heparin-binding polypeptides to be administered inaccordance with the present invention are known in the art, and so aretheir therapeutically effective doses. The determination of theeffective dose for new functional derivatives may require some routineexperimentation, but in all instances, the determination of atherapeutically effective dose is well within the skill in the art.

The terms "DNA sequence encoding", "DNA encoding" and "nucleic acidencoding" refer to the order or sequence of deoxyribonucleotides along astrand of deoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide chain. The DNAsequence thus codes for the amino acid sequence.

II. Preferred Embodiments

In addition to commercially available purified native heparinpreparations (for example from intestinal mucosa), compounds which arecapable of inhibiting the binding of a heparin-binding protein tolow-affinity heparin-like substances (binding sites) on cell surface orin the extracellular matrix include other polyanionic compounds, such asdextran sulfate, heparan sulfate, pentosan (poly) sulfate, hyaluronateand heparin fragments. Heparin fragments may be obtained from a libraryof heparin-derived oligosaccharides that have been fractionated bygel-permeation chromatography. The preparation of affinity-fractionated,heparin-derived oligosaccharides was reported by Ishihara et al., J.Biol. Chem. 268, 4675-4683 (1993). These oligosaccharides were preparedfrom commercial porcine heparin following partial depolymerization withnitrous acid, reduction with sodium borohydride, and fractionation bygel permeation chromatography, The resulting pools of di-, tetra-,hexa-, octa-, and decasaccharides were sequentially applied to anaffinity column of human recombinant bFGF covalently attached toSepharose 4B, and were further fractionated into subpools based on theirelution from this column in response to gradients of sodium chloride.This resulted in five pools, designated Hexa-1 to Hexa-5, the structuresand biological activities of which were further evaluated. The structureof Hexa-5C and its 500-MHz NMR spectrum are shown in FIG. 4 of Tyrell etal., J. Biol. Chem. 268, 4684-4689 (1993). This hexasaccharide has thestructure [IdoA(2-OSO₃)α1-4GlcNSO₃ (6-OSO₃)α1-4]₂ IdoA(2-OSO₃)α1-4AMan_(R) (6-OSO₃). All heparin-derived oligosaccharidesdiscussed above, as well as other heparin-like oligosaccharides aresuitable for and can be used in accordance with the present invention.It is preferred, however, to use hexasaccharides and polysaccharides ofhigher unit size (e.g. hepta-, octa-, nona- and decasaccharides) topractice the present invention. Furthermore, heparin-derived orheparin-like oligosaccharides with a large net negative charge, e.g. dueto a high degree of sulfation, are used with advantage. In therapeuticapplications, the use of specific heparin fragments has a distinctadvantage over that of intact heparin. Heparin is an antithrombotic andmay be toxic, especially in higher concentrations. By using smallerheparin fragments, the antithrombin-3 (AT3) binding site of heparin canbe effectively engineered out, and thus the fragments are expected to bedevoid of antithrombotic properties.

Whereas the use of polyanionic compounds in conjunction withnative-sequence heparin-binding proteins is within the scope of thepresent invention, the methods and compositions herein are equallysuitable for the purification, detection and administration of anyheparin-binding protein as hereinabove defined, including nativeheparin-binding proteins, their functional derivatives, and inhibitorsof their biological actions.

In a preferred embodiment, the heparin-binding protein is HGF, morepreferably native huHGF or a functional derivative or inhibitor thereof.HGF variants are, for example, disclosed in U.S. Pat. No. 5,316,921 andU.S. Pat. No. 5,328,837. As it has been shown that the receptor bindingdomain is contained within the finger and Kringle 1 (K1) regions of thenative huHGF molecule, in addition to the heparin-binding site(s), theHGF variants preferably contain a functional finger and K1 region. Inanother preferred group of HGF variants a functional Kringle 2 (K2)region is additionally present. We have experimentally found that huHGFvariants composed of the finger, K1 and K2 domains of native huHGFretain the ability to bind heparin, i.e. contain at least oneheparin-binding site. Single-chain HGF variants, which are resistant toproteolytic cleavage by trypsin-like proteases at the one-chain totwo-chain cleavage site between Arg494 and Val495 of native huHGF areable to bind the HGF receptor but substantially lack biological activity(i.e. they are HGF inhibitors). Such variants preferably contain singleor multiple amino acid substitutions, insertions and/or deletions at oradjacent to amino acid positions 493, 494, 495 and 496 of the nativehuHGF amino acid sequence. A preferred alteration is the replacement ofarginine at amino acid position 494 with any other amino acid,preferably glutamic acid, aspartic acid or alanine. Alterations thatpotentially increase the receptor binding capacity of native huHGF are,for example, in the amino acid region corresponding to a potentialserine protease active site. This region includes amino acids Q534, Y673and V692 in the native huHGF amino acid sequence. The replacement ofthese amino acids with any other amino acid, and preferably with aminoacids of different size and/or polarity is believed to further improvethe receptor binding properties of huHGF.

In another preferred embodiment, the heparin binding protein is aselectin or a functional derivative thereof. The selectin preferably isL-selectin (U.S. Pat. No. 5,098,833 issued 24 Mar. 1992) or E-selectin.Functional derivatives of selectins are also known are and, for example,disclosed in U.S. application Ser. No. 07/879,036 filed 30 Apr. 1992,now abandoned. If the functional derivative of a selectin is an aminoacid sequence variant, it will ordinarily have a lectin domain the aminoacid sequence of which is substantially homologous (more than about 80%based on complete amino acid identity, ignoring insertions anddeletions) to that of a native selectin molecule. The amino acidresidues identified as primarily involved with the recognition of thecarbohydrate ligands of selectins are a relatively small patch on thesurface of the selectin lectin domain near the antiparallel beta sheetformed by the disulfide-linked N- and C-termini and the adjacentdisulfide-linked loop formed by the two internal cysteines. Asignificant increase in ligand binding has been described for selectinvariants having a amino acid substitution at position 8. In particular,the substitution of alanine for glutamic acid (a charged residue) atposition 8 of E-selectin, and the mutation of lysine to alanine at thissite in L- and P-selectin was found to significantly enhance ligandrecognition. The use of selectin variants containing one or more aminoacid alterations in the amino acid regions 7-9, 43-48, 82-86, 90-109,and at amino acid positions 111 and 113 is specifically within the scopeof the present invention.

The heparin-binding protein and the binding inhibitory compound, whichis typically a polyanionic compound, can be incorporated in the samepharmaceutical formulation or may be otherwise coadministered, whereincoadministration includes simultaneous and tandem administrations ineither order, provided that the polyanionic compound is administered ata time when no significant amount of the heparin-binding compound hasbeen removed from the blood stream. This time is preferably not laterthan the time by which about 10% of the original dose administered hasbeen removed from the circulation.

Dosages of the heparin-binding proteins and their functional derivativesadministered in accordance with the present invention may vary dependingon the actual compounds used, and on the use envisioned. A typicaleffective dose of HGF in rat experiments is about 250 μg/kg administeredas an intravenous bolus injection. Interspecies scaling of dosages canbe performed in a manner known in the art, e.g. as disclosed by Mordentiet al., Pharmaceut. Res. 8, 1351 (1991). The therapeutically effectivedaily dose of native huHGF typically is about 0.01 to 100 mg in humanpatients, which can be administered as a single dose or in the form ofmultiple doses. Typical doses and formulations of L-selectin are, forexample, disclosed in U.S. Pat. No. 5,098,833, issued 24 Mar. 1992.

In general, suitable ingredients for use in pharmaceutical compositionsare described in Remington's Pharmaceutical Sciences, 16th edition,1980, Mack Publishing Co., edited by Oslo et al.

The ratio of the heparin-binding protein and the polyanionic compound,such as heparin or heparin-derived oligosaccharides, is believed to benot critical. As a general proposal, the polyanionic compound is used inmolar excess as compared to the heparin binding protein. For HGF, thiscan be an about 10-fold mass excess. The determination of the optimumratio for each combination of heparin binding proteins and polyanioniccompounds is well within the skill in the art. The actual dose of thepolyanionic compound used in association with a heparin-binding proteinalso depends on the purpose of administration. For example, as shown inExample 6, the bioavailability of HGF is efficiently increased bydextran sulfate administered in a 1:1-1:10, preferably 1:1-1:2HGF/dextran sulfate molar ratio, preferably using an about 2 to 5mg/kg/day dose of dextran sulfate. Similar ratios and total doses forother heparin-binding protein/polyanionic compound pairs can bedetermined experimentally.

The polyanionic compounds used in accordance with the present inventioncan be incorporated into pharmaceutical compositions known for theadministration of the corresponding heparin-binding proteins.

The coadministration in accordance with the present inventionspecifically includes the possibility of administering more than oneheparin-binding protein and more than one polyanionic compound, as itmight be desirable for any given application.

Further details of the invention are illustrated in the followingnon-limiting examples.

EXAMPLE 1 (FIG. 1) The effect of heparin on the biological activity ofHGF.

The effect of heparin on the biological activity of single chain HGF wasmeasured by comparing the ability of HGF alone or in the presence ofheparin to induce DNA synthesis in rat hepatocytes in primary culture(Lokker et al., EMBO J. 11: 2503-2510 (1992)). Hepatocytes were isolatedaccording to published perfusion techniques (Garrison and Haynes, J.Biochem. 250: 2269-2777 (1975)) and resuspended at 1×10⁵ cells/ml inserum-free bioassay medium containing Williams media, insulin,transferrin, glutamine, BSA and antibiotics. Hepatocytes were thenincubated in 96-well plates in the presence of duplicate serialdilutions of rhuHGFo To some wells, heparin was added at 0.1, 1.0, or 10μg/mL. After 48 hr incubation at 37° C., 0.5 mCi[³ H]TdR (15 Ci/mmol,Amersham) was added to each well and incubated for an additional 16 hr.Cells were harvested on filter papers, which were washed, dried andcounted in a Beckman counter after addition of scintillation fluid.Relative radioactivity incorporated into hepatocytes is plotted as afunction of HGF concentration.

EXAMPLE 2 (FIG. 2) The effect of heparin on the detection of HGF in anELISA.

Either single chain (panel a) or two chain (panel B) HGF concentrationswere measured using a buffer-based immunoassay in the presence ofvarying concentrations of heparin (0, 0.1, and 10 μg/mL). HGF andheparin were diluted in ELISA diluent containing PBS, 0.1% BSA, and0.05% Tween-20. The ELISA assay used monoclonal antibody A.3.1.2 as acoat antibody and B.4.3 conjugated to horse radish perioxidase fordetection.

EXAMPLE 3 (FIG. 3) The effect of heparin on the pharmacokinetics of HGFin vivo.

New Zealand white rabbits (n=3/group) were injected with an iv bolusdose of HGF (0.2 mg/kg) in the presence and absence of 200 unitsheparin/kg. Blood samples were harvested at various times followinginjection. HGF concentrations in rabbit plasma were measured using aplasma-based HGF specific ELISA. The weight normalized clearance, CL,initial volume of distribution, V₁, steady state volume of distribution,Vss, mean residence time, MRT, and half-life, t_(1/2) are presented.

EXAMPLE 4 (FIG. 4) The effect of polyanions on the bioavailability ofHGF after subcutaneous administration.

Rats (n=6/group) received subcutaneous injections of HGF (500 μg/kg) inthe presence or absence of a 10-fold mass excess of various polyanions.HGF was allowed to incubate with the respective polyanion for 60 min atroom temperature prior to injection. Rat blood samples were removed 60min following HGF administration. HGF concentrations in blood weremonitored using a plasma based ELISA.

EXAMPLE 5 (FIGS. 5A-5B) The effect of heparin on the hepatic clearanceof [¹²⁵ I]HGF.

The effect of heparin on the hepatic clearance of HGF was studied in asingle pass liver perfusion (SPLP) experiment. Approximately 10 ng/ml[¹²⁵ ]HGF was infused via the portal vein into a rat liver in thepresence or absence of heparin. The amount of HGF that was not bound bythe liver after a single pass was determined and expressed as percent ofinfused dose. Both total and TCA precipitable radioactivity weremonitored.

EXAMPLE 6 Increase of bioavailability of HGF/SF by coadministration ofdextran sulfate MATERIALS AND METHODS

Animals: Male C3H/HeJ mice were obtained from The Jackson Laboratory(Bar Harbor, Me.) at 6-8 weeks of age (body weight=23-27 g) and allowedto acclimatize for at least one week before use. Food (Formulab Chow,Purina Miles, Inc.) and water were provided ad libitum, room temperaturewas maintained at 72° F., and the light:dark cycle as 12h:12h. Allexperiments were approved and monitored by the Genentech Animal Care andUse Committee, following guidelines established by the NationalInstitutes of Health.

Recombinant human HGF/SF was produced at Genentech, Inc. (South SanFrancisco, Calif.) and formulated in 20 mM TrisHCl (pH 7.5), 500 mMNaCl. Endotoxin levels were <2 EU/mg, as determined in the limuluslysate assay. Dextran sulfate (sodium salt, average MW=5,000) waspurchased from Sigma Chemical Co. (St. Louis, Mo.). HGF/SF and/ordextran sulfate were diluted with 0.9% pyrogen-free saline (McGaw, Inc.,Irvine, Calif.), sterilized using 0.2 mm filters (Millipore ProductsDivision, Bedford, Mass.), and then loaded under sterile conditions intoAlzet Model 1003D osmotic minipumps (Alza Corporation, Palo Alto,Calif.), which have a nominal delivery rate of 24 ml/day. In someexperiments in which the HGF/SF concentration in the pump load was lessthan 1 mg/ml, bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.)was included at 1 mg/ml. Pumps were implanted intraperitoneally througha midline incision, using aseptic technique. Anesthesia was induced withketamine (Ketaset, Aveco Co., Iowa City, Iowa, 75 mg/kg) and xylazine(Rompin, Rugby Laboratories, Inc., N.Y., 7.5 mg/kg). The animals wereallowed to recover on heating pads and returned to group caging. Pumpswere always implanted in the late afternoon and experiments wereterminated in the morning three days (˜66 hours) later so that the pumpswere allowed to deliver HGF/SF as long as possible, but samples werecollected before the pumps were exhausted.

Analyses: Body weight was recorded, then trunk blood was collected bycardiac puncture under ketamine-xylazine anesthesia. One aliquot wasadded to an EDTA microtainer tube and the remainder was allowed to clot.Serum and plasma samples were stored at -70° C. Wet weights of liver,kidneys, and spleen were recorded.

For histological analysis, portions of tissue were fixed in 10% neutralbuffered formalin and paraffin sections were stained with hematoxylinand eosin. Mitotic figures were scored in 20 fields under 20× objective.Fields were selected blindly and those in which hepatic parenchyma didnot occupy the entire field --for example, fields that contained largeveins or were transected by the edge of the section--were excluded. Theanalysis was performed by an individual blinded to the treatment groups.

DNA was quantitated after extraction and precipitation withtrichloroacetic acid (Burton, K. A., Biochem. J. 62, 315-323 [1956]).Plasma concentrations of recombinant human HGF were measured using aspecific sandwich ELISA, as described previously (Mendenhall et al.,Hepatol. submitted, 1994). Serum biochemistries were measured on aMonarch Model 2000 Model 761 Microcentrifugal Chemistry Analyzer(Instrumentation Laboratories, Lexington, Mass.).

Statistical Methods: Animals were randomly assigned to treatment groupsand all experiments were performed in a blinded manner. The results areexpressed as mean ± SEM of 5-8 animals. Data were analyzed by analysisof variance, followed by Duncan's multiple range test, except thathistological scoring was analyzed nonparametrically, using theMann-Whitney U test.

RESULTS

HGF/SF was loaded into osmotic minipumps alone or in combination withdextran sulfate. We had previously found that bioavailability of HGF/SFafter subcutaneous or intraperitoneal injection was markedly enhancedwhen HGF/SF was premixed with dextran sulfate. In those studies, a10-fold mass excess of dextran sulfate was used. In Experiment 1, theconcentration of HGF/SF in the pumps was 2.4 mg/ml, which produced adaily dose of 2.4 mg/kg, and the HGF/SF:dextran sulfate ratio was 1:10,as used in the injection studies. We also tested a 1:2 ratio. The pumps:were implanted in the late afternoon and the animals were killed in themorning 3 days later (˜66 hours). As shown in the upper portion of Table1, plasma levels of immunoactive HGF/SF in Experiment 1 were increasedslightly but significantly when HGF/SF alone was infused, compared tovehicle-treated controls, but there was no effect on wet liver weight.Dextran sulfate alone also had no effect. When HGF/SF was combined witha 2-fold mass ratio of dextran sulfate, plasma HGF levels were increasedfurther than with HGF alone. Moreover, there was a significant increasein liver wet weight. There was no effect on serum HGF or liver wetweight when the HGF/SF:dextran sulfate ratio was 1:10.

At the concentrations of dextran sulfate needed to achieve the 1:10ratio, the material was extremely viscous. Therefore, in Experiment 2,we tried HGF/SF:dextran sulfate ratios of 1:2 and 1:1, with the dailyHGF/SF dose again fixed at 2.4 mg/kg. As shown in the lower portion ofTable 1, this experiment confirmed the increases in serum HGF/SF levelsand liver weight after infusion of HGF/SF with dextran sulfate at a 1:2mass ratio. Similar effects were observed at a 1:1 ratio and this ratiowas used in all subsequent experiments.

In Experiment 3, we tested several different doses of HGF/SF (0.27, 0.8or 2.4 mg/kg/d), with the HGF/SF:dextran sulfate ratio held Constant at1:1. Consistent with earlier experiments, liver weight was increased inthe 2.4 mg/kg/d group (6.9±0.1% body Wt for HGF/SF-treated animals, vs5.5±0.2 for controls, P<0.01), but there was no effect at the two lowerHGF/SF doses (data not shown). In Experiment 4, HGF/SF doses of 1.2,2.4, and 4.8 mg/kg/d were tested, again with a fixed 1:1 HGF/SF:dextransulfate ratio. One control group received pumps containing only thebuffer vehicle. A second control group received dextran sulfate alone at4.8 mg/kg/d. There were no controls treated with lower doses of dextransulfate alone. As shown in FIG. 6, serum HGF/SF levels were very low incontrols that received neither HGF/SF nor dextran sulfate, and inanimals given dextran sulfate alone, but were increased in adose-dependent manner when the pumps contained increasing concentrationsof HGF/SF. Liver weight was increased markedly in both the 2.4 and 4.8mg/kg/d dose groups compared to controls, and these two groups were notdifferent from each other. When liver wet weight was plotted againstplasma HGF/SF in individual animals, liver wet weight increased asplasma HGF/SF levels rose above baseline but reached a plateau (FIG. 7).

We repeated these studies several times and as can be seen in Table 2,enlargement of the liver, measured either as absolute liver weight ornormalized to body weight, was observed in every experiment in which theHGF/SF dose was 2.4 mg/kg/d or greater. The-average increase in size was27±8 %. In contrast, there was no effect on the weight of kidney orspleen in any experiment (Table 2).

On gross examination, livers from control and HGF/SF treated animalsdiffered only in size. Histologically, the livers induced to grow byHGF/SF-dextran sulfate looked normal, except that mitotic figures, whichwere rarely found in sections of control liver, were frequently seen inHGF/SF-treated animals (FIGS. 8A-8C ). To quantify this effect, thenumber of mitotic figures in 20 randomly-selected fields were counted.The results are shown in Table 3. An average of ˜1 mitotic figure wasfound throughout all 20 fields from control livers and from liversexposed to either dextran sulfate alone or 1.2 mg/kg/d HGF/SF. Incontrast, approximately 30 figures were found in the 20 fields: fromlivers exposed to higher doses of HGF/SF, or 1-2 in each field. Nomitotic figures were observed in bile duct epithelium. Total liver DNAincreased by 21% in Experiment 5 and by 34% in Experiment 6 (Table 3),although this effect did not reach statistical significance inExperiment 5 (P=0.14).

Serum samples from HGF/SF-treated animals were frequently lipemic, whichled us to measure a panel of serum biochemistries (Table 4). Consistentwith the lipemic appearance, serum triglycerides and cholesterol weremarkedly elevated in a dose-dependent manner. In addition, total serumprotein and serum albumin were increased. Conversely, alkalinephosphatase was reduced by HGF/SF. No effect on either ALT (Table 4) orAST (not shown) was detected in any experiment.

DISCUSSION

In the course of studying the effects of HGF/SF in animal models, wefound that HGF/SF is very poorly absorbed into the blood stream aftersubcutaneous injection. Bioavailability was better after intraperitonealinjection, but still poor. Consistent with these observations, studiesthat have shown activities of HGF/SF in vivo have used intravenousdelivery (Fujiwara et al., Hepatol. 18, 1443-1449 [1993]; Ishiki et al.,Hepatol. 16, 1227-1235 [1992]; Roos et al., Endocrinol. 131, 2540-2544[1992]). Because HGF/SF is cleared very rapidly (Appasamy et al., Lab.Invest. 68, 270-276 [1993]; Zionchem. et al., Endocrin. 134, 1879-1887[1994]), serum levels are elevated for only brief periods of timefollowing each iv injection. The data presented here show thatintraperitoneal infusion of HGF/SF combined with dextran sulfateprovides sustained elevations in circulating HGF/SF levels.

The absolute values for plasma HGF/SF varied among experiments. As anextreme example, the same dose of HGF/SF and dextran sulfate (2.4mg/kg/d and 4.8 mg/kg/d, respectively) produced plasma levels of 28.3ng/ml in Experiment 1 and 7.1 ng/ml in Experiment 2 (see Table 1). Thesource of this variability is unclear but seems to be related more tointer-experiment factors than interassay variability because similarvalues were obtained when representative samples were re-run within asingle ELISA. It is for this reason that the correlation between plasmaHGF/SF and liver weight shown in FIG. 7 was limited to data from asingle experiment.

The rationale for the use of dextran sulfate is that in a separateseries of experiments, we found that premixing of HGF/SF with solubleheparin greatly enhanced its bioavailability from subcutaneous andintraperitoneal sites (unpublished observation). This effect was notspecific to heparin and was also observed with other sulfatedpolysaccharides including pentosan polysulfate, hyaluronate, and dextransulfate. Dextran sulfate was chosen arbitrarily for the infusionstudies. At a 1:10 HGF/SF:dextran sulfate ratio the solution wasextremely viscous and probably could not get out of the infusion pump.However, ratios of 1:1 or 1:2 enhanced the effectiveness of HGF/SFinfusions. Although the mechanism is not known, we hypothesize that,because HGF/SF binds heparin strongly (Nakamura et al., Proc. Natl.Acad. Sci. USA 83, 6489-6493 [1986]), its absorption from subcutaneousand intraperitoneal sites is low due to trapping by interaction withheparin sulfate proteoglycans in the extracellular matrix. We thereforespeculate that sulfated polysaccharides saturate the heparin bindingregions on HGF/SF, thereby preventing interaction with matrixcomponents. Heparin also decreases the clearance of HGF/SF, and sulfatedpolysaccharides enhance hepatocyte responses to low doses of HGF/SF.

Using this delivery system, we have been able to induce growth of liverin an otherwise intact animal. While this work was in progress, Fujiwaraet al. reported that HGF stimulated liver growth in normal rats andenhanced the amount of liver regenerated after partial hepatectomy(Fujiwara et al., Hepatol. 18, 1443-1449 [1993]). In those studies, HGFwas injected intravenously every 2 hours for 10 hours. Each injectionwas 10 μg/100 g body weight, a total dose 500 μg/kg. This dose is lowerthan the daily dose we found to be necessary to induce comparable livergrowth in our studies, but it compares favorably when one considers thatin our studies the material was delivered by continuous extravascularinfusion while in those of Fujiwara et al., it was delivered directlyinto the bloodstream. To our knowledge, these are the only two reportsthat a growth factor has been able to induce growth of normal liver invivo, despite the fact that a large number of proteins can stimulatehepatocyte DNA synthesis in vitro.

A major mechanism for the HGF/SF-induced increase in wet liver weightappears to be stimulation of hepatocyte proliferation. Mitotic figures,which are rarely observed in histological sections from control liver,were abundant in livers exposed to HGF/SF. Mitoses were found randomlydistributed throughout the hepatic lobule, with no apparent associationwith vascular elements. Although HGF/SF has been reported to stimulateDNA synthesis in nonparenchymal cells and bile duct epithelial cells invitro (Joplin et al., J. Clin. Invest. 90, 1284-1289 [1992]), weobserved no mitotic figures in bile duct epithelial cells. However, thenumber of such cells in any section is very small compared to the numberof hepatocytes, so a modest amount of proliferation in this compartmentwould be difficult to detect histologically.

Hepatic DNA content was elevated in the HGF/SF-treated animals in bothexperiments in which it was measured, although this did not reachstatistical significance in Experiment 5. The difference in DNA levelsbetween these two experiments are due to fact that polypropylene tubeswere used for the DNA assay in Experiment 5 while glass tubes were usedfor Experiment 6; dissolution of the pellet after TCA precipitation waseasier in glass tubes, giving better extraction efficiency.

There also appears to be a component of the increase in liver wet weightthat is not related to proliferation. For example, a dose of 1.2 mg/kg/dclearly had no effect on mitotic activity (Table 3), but causedsignificant increases in liver wet weight (Table 3), serum lipids (Table4), and a significant decrease in alkaline phosphatase (Table 4). Othershave reported striking effects of HGF/SF on the morphology of severalcell types (Bhargava et al., EXS 65, 341-349 [1993]; Li et al., InVitro. Cell Dev. Biol. 28A, 364-368 [1992]; Uehara et al., J. Cell.Biol. 117, 889-894 [1992]), suggesting that it may have trophic as wellas proliferative actions in target tissues. Our data are consistent withsuch a trophic action in the liver.

Expression of HGF/SF in the kidney has been shown to increase followingunilateral nephrectomy, which has raised the suggestion that HGF/SF isimportant in compensatory renal hypertrophy (Nagaike et al., J. Biol.Chem. 266, 22781-22784 [1991]). However, we consistently found no effecton kidney size, despite the fact that liver size was increased in everyexperiment. Infusions of longer duration have also shown no effects onkidney size (unpublished observation). The sensitivity of the kidney toHGF/SF may be regulated locally in different physiological states.

Lastly, we observed that HGF/SF-induced liver growth was associated withchanges in serum biochemistry, the most striking of which was lipemia.Total protein and serum albumin were also markedly elevated. The reasonthat alkaline phosphatase was reduced is unknown. Nonetheless, theseresults indicate that HGF/SF has major effects on hepatic function, aswell as size.

In addition, we have shown that normal liver can be induced to grow bytreatment with intraperitoneally administered HGF/SF in the absence ofany other perturbation or stimulation. The increase in liver size wasaccompanied by marked increases in proliferative activity of hepatocytesand in increases in the level of serum protein and lipid.

In conclusion, we have shown that intraperitoneal infusion of HGF/SF incombination with dextran sulfate is an effective mechanism for providingsustained elevations in circulating HGF/SF.

                  TABLE 1                                                         ______________________________________                                        Effect of Infusion of HGF/SF with Dextran Sulfate on Plasma                   HGF and Liver Weight.                                                                                              Liver                                          Dose                  Plasma   Weight                                   Experi-                                                                             (mg/kg/d)             HGF/SF   (% Body                                  ment  HGF/SF   DexSO.sub.4                                                                            Ratio (ng/ml)  Wt)                                    ______________________________________                                        1     0        0              0.9 ± 0.4                                                                           5.8 ± 0.1                                 2.4      0        0      5.9 ± 2.0*                                                                         5.7 ± 0.2                                 0        4.8            0.5 ± 0.1                                                                           5.9 ± 0.2                                 2.4      4.8      1:2    28.3 ± 10.7*                                                                        7.1 ± 0.5*                               0        24.0           0.6 ± 0.2                                                                           5.5 ± 0.1                                 2.4      24.0      1:10 1.1 ± 0.5                                                                           5.5 ± 0.1                           2     0        0              0.6 ± 0.1                                                                           5.4 ± 0.1                                 0        4.8            0.6 ± 0.1                                                                           5.8 ± 0.2                                 2.4      4.8      1:2    7.1 ± 1.8*                                                                          6.4 ± 0.2*                               0        2.4            0.5 ± 0.1                                                                           5.4 ± 0.2                                 2.4      2.4      1:1    6.0 ± 2.3*                                                                          6.2 ± 0.1*                         ______________________________________                                         Osmotic minipumps filled to deliver the indicated doses of HGF/SF and         dextran sulfate were implanted intraperitoneally in intact mice. Plasma       HGF and liver wet weight were determined on the third day of infusion.        Values are means ± SEM, n = 5-6 per group.                                 *Different from untreated control group in the same experiment (P < 0.01)

                                      TABLE 2                                     __________________________________________________________________________    Effects of 3-day infusions of HGF/SF and dextran sulfate on wet weights       of liver, kidney, and                                                         spleen.                                                                       Exp  Dose (mg/kg/d)                                                                          Liver Weight        Kidney Wt (Pair)                                                                       Spleen Wt                         No.  HGF/SF                                                                             DexSO.sub.4                                                                        Absolute (g)                                                                         (% Body Wt)                                                                          % Increase                                                                          (% Body Wt)                                                                            (% Body Wt)                       __________________________________________________________________________    2    0    2.4  1.22 ± 0.04                                                                       5.4 ± 0.2 1.60 ± 0.07                                                                         0.44 ± 0.01                         2.4  2.4   1.42 ± 0.03*                                                                      6.2 ± 0.1*                                                                       15%   1.60 ± 0.11                                                                         0.41 ± 0.03                    3    0    2.4  1.30 ± 0.05                                                                       5.7 ± 0.1 1.70 ± 0.05                                                                         0.41 ± 0.02                         2.4  2.4   1.54 ± 0.03*                                                                      6.9 ± 0.1*                                                                       21%   1.68 ± 0.03                                                                         0.40 ± 0.02                    4    0    4.8  1.29 ± 0.05                                                                       5.7 ± 0.2 1.62 ± 0.03                                                                         0.38 ± 0.01                         2.4  2.4   1.69 ± 0.07*                                                                      7.5 ± 0.2*                                                                       32%   1.63 ±  0.03                                                                        0.42 ± 0.02                         4.8  4.8   1.63 ± 0.02*                                                                      7.8 ± 0.2*                                                                       37%   1.59 ± 0.01                                                                         0.41 ± 0.01                    5    0    4.8  1.11 ± 0.03                                                                       5.4 ± 0.1 ND       ND                                     4.8  4.8   1.45 ± 0.06*                                                                      7.2 ± 0.2*                                                                       33%   ND       ND                                6    0    2.4  1.47 ± 0.05                                                                       5.9 ± 0.1 1.77 ± 0.02                                                                         0.35 ± 0.02                         2.4  2.4   1.85 ± 0.07*                                                                      7.4 ± 0.3*                                                                       25%   1.82 ± 0.03                                                                         0.36 ± 0.02                    Overall                      27 ± 8                                        __________________________________________________________________________     Adult mice were infused intraperitoneally with the indicated doses of         HGF/SF and dextran sulfate. Three days later organ weights were               determined. Values are means ± SEM, n = 5-7 per group. ND = Not            Determined.                                                                   *P < 0.01 compared to dextran sulfate alone within the same experiment.  

                                      TABLE 3                                     __________________________________________________________________________    Mitotic activity and DNA content liver after infusion                         of HGF/SF.                                                                    Exp                                                                              Dose (mg/kg/d)                                                                          Liver Wt                                                                              Mitoses Liver DNA                                        No.                                                                              HGF/SF                                                                             DexSO.sub.4                                                                        (% Body Wt)                                                                           per 20 fields                                                                         (mg/100 g BW)                                    __________________________________________________________________________    4  0    0    5.6 ± 0.2                                                                          0.8 ± 0.5                                                                          ND                                                  0    4.8  5.7 ± 0.2                                                                          0.3 ± 0.2                                                                          ND                                                  1.2  1.2   6.2 ± 0.1*                                                                        0.8 ± 0.4                                                                          ND                                                  2.4  2.4   7.5 ± 0.2**                                                                        37.0 ± 19.6**                                                                     ND                                                  4.8  4.8   7.8 ± 0.2**                                                                        54.0 ± 6.9**                                                                      ND                                               5  0    4.8  5.4 ± 0.1                                                                          1.5 ± 0.8                                                                          10.7 ± 0.6                                       4.8  4.8  7.2 ± 0.2                                                                           29.8 ± 9.8**                                                                      12.9 ± 1.2                                    6  0    2.4  5.9 ± 0.1                                                                          0.8 ± 0.5                                                                          13.7 ± 0.7                                       2.4  2.4   7.4 ± 0.3**                                                                        30.3 ± 14.9**                                                                      18.3 ± 1.1**                                 __________________________________________________________________________     Adult mice were infused intraperitoneally with the indicated doses of         HGF/SF and dextran sulfate for three days. A portion of liver was then        fixed and sections were stained with hematoxylin and eosin. The number of     mitotic figures in 20 fields under a 20x objective was counted in a           blinded manner. DNA was extracted for an additional portion of liver.         Values are means ± SEM, n = 5-7 per group. ND = Not Determined.            *P < 0.05, **P < 0.01 compared to control group within the same               experiment.                                                              

                                      TABLE 4                                     __________________________________________________________________________    Effects of 3-day Infusions of HGF/SF and dextran sulfate on serum             biochemistry.                                                                                                   Total                                       Exp.                                                                             Dose (mg/kg/d)                                                                          Liver Wt                                                                              Triglycerides                                                                        Cholesterol                                                                         Protein                                                                             Albumin                                                                             Alk. Phos.                                                                          ALT                       No.                                                                              HGF/SF                                                                             DexSO.sub.4                                                                        (% Body WT)                                                                           (mg/dl)                                                                              (mg/dl)                                                                             (g/dl)                                                                              (g/dl)                                                                              (U/L) (IU/L)                    __________________________________________________________________________    3  0    0    5.7 ± 0.1                                                                          104 ± 9                                                                           ND    ND    ND     85 ± 2                                                                          50.2 ± 15.7               0    2.4  5.7 ± 0.1                                                                          129 ± 10                                                                          ND    ND    ND     77 ± 4                                                                          78.4 ± 19.6               2.4  2.4  6.9 ± 0.1*                                                                         287 ± 63**                                                                        ND    ND    ND     52 ± 3**                                                                        55.5 ± 18.6            4  0    0    5.6 ± 0.2                                                                          134 ± 29                                                                          151 ± 7.sup.a                                                                    ND    ND     70 ± 3.sup.a                                                                    45.3 ± 8.3                0    4.8  5.7 ± 0.2                                                                          102 ± 23                                                                          147 ± 5.sup.a                                                                    ND    ND     71 ± 1.sup.a                                                                    37.2 ± 5.6                1.2  1.2  6.2 ± 0.1*                                                                         155 ± 18                                                                          224 ± 3.sup.b                                                                    ND    ND     54 ± 3.sup.b                                                                    45.8 ± 9.0                2.4  2.4  7.5 ± 0.2**                                                                        344 ± 81*                                                                         310 ± 16.sup.c                                                                   ND    ND     48 ± 3.sup.c                                                                    37.0 ± 4.8                4.8  4.8  7.8 ± 0.2**                                                                        751 ± 92**                                                                        406 ± 16.sup.d                                                                   ND    ND     29 ± 9.sup.d                                                                    48.0 ± 10.7            5  0    4.8  5.4 ± 0.1                                                                           79 ± 9                                                                           133 ± 3                                                                          4.9 ± 0.1                                                                        3.0 ± 0.1                                                                        107 ± 7                                                                          55.0 ± 7.5                4.8  4.8  7.2 ± 0.2**                                                                        350 ± 82**                                                                        275 ± 30**                                                                       7.3 ± 0.5**                                                                      3.8 ± 0.3*                                                                        72 ± 9**                                                                        48.0 ± 11.7            6  0    2.4  5.9 ± 0.1                                                                          113 ± 20                                                                          110 ± 4                                                                          4.5 ± 0.1                                                                        2.7 ± 0.1                                                                         75 ± 3                                                                          68.2 ± 20.7               2.4  2.4  7.4 ± 0.3*                                                                         271 ±  102                                                                        208 ± 22**                                                                       5.9 ± 0.3**                                                                      3.5 ± 0.1**                                                                       48 ± 4**                                                                        78.0                      __________________________________________________________________________                                                        ± 32.6                  Adult mice were infused intraperitoneally with the indicated doses of         HGF/SF and dextran sulfate for three days, then serum biochemistries were     measured. Values are means ± SEM, n = 5-7 per group. ND = Not              Determined.                                                                   *P < 0.05, **P < 0.01 compared to control group within the same               experiment. In Experiment 4, groups marked with different letters are         different from each other at P < 0.01. The large error in the HGF/SF grou     in Experiment 6 was due primarily to one animal with a value of 759 mg/dl                                                                              

The entire disclosures of all citations cited throughout thespecification, and the references cited therein, are hereby expresslyincorporated by reference.

We claim:
 1. A method of extending the plasma half-life of a selectincomprising coadministering a therapeutically effective amount of saidselectin with a therapeutically acceptable compound capable ofinhibiting the binding of said selectin to a low affinity heparin-likebinding site on the surface of cells of an organ perfused with blood. 2.The method of claim 1 wherein said cells are hepatic cells.
 3. Themethod of claim 1 wherein said cells are renal cells.
 4. The method ofclaim 1 wherein said selectin is L-selectin.
 5. The method of claim 4wherein said L-selectin is pnHR, LEC-CAM-1, LAM-1, gp90^(MEL),GP100^(MEL), gp110^(MEL), MEL-14 antigen, Leu-8 antigen, TQ-1 antigen,or DREG antigen.
 6. The method of claim 1 wherein the binding inhibitorycompound is a polyanionic compound.
 7. The method of claim 6 whereinsaid polyanionic compound is a dextran, heparin or a functionalderivative thereof.
 8. The method of claim 7 wherein said functionalderivative is heparan sulfate or dextran sulfate.
 9. The method of claim7 wherein said functional derivative is an oligosaccharide which is afunctional derivative of heparin.
 10. The method of claim 1 wherein saidselectin and the binding inhibitory compound are administeredconcurrently.
 11. The method of claim 1 wherein said selectin and thebinding inhibitory compound are incorporated in the same pharmaceuticalcomposition.
 12. The method of claim 1 wherein said selectin isE-selectin.
 13. The method of claim 12 wherein said E-selectin isLEC-CAM-2, LECAM-2, or ELAM-1.
 14. The method of claim 1 wherein saidselectin is P-selectin.
 15. The method of claim 14 wherein saidP-selectin is LEC-CAM- 3, LECAM- 3, GMP- 140, or PADGEM.
 16. A methodfor improving the therapeutic potency of a selectin comprisingcoadministering a therapeutically effective amount of said selectin witha therapeutically acceptable polyanionic compound.
 17. The method ofclaim 16 wherein said polyanionic compound is heparin or a functionalderivative thereof.
 18. The method of claim 17 wherein the functionalderivative is a heparin-derived oligosaccharide.
 19. A method ofimproving the bioavailability of a subcutaneously or intraperitoneallyadministered selectin comprising coadministering said selectin with atherapeutically acceptable compound capable of inhibiting theinteraction of said selectin with heparin or a substance which is afunctional derivative of heparin in an extracellular matrix.
 20. Themethod of claim 19 wherein said therapeutically acceptable compound is apolyanionic compound.
 21. The method of claim 20 wherein saidpolyanionic compound is a dextran, heparin or a functional derivativethereof.
 22. The method of claim 21 wherein said functional derivativeis dextran sulfate.
 23. A method for preventing the inactivation of aselectin by a blood protein, which comprises coadministering aneffective dose of said selectin with a polyanionic compound.
 24. Apharmaceutical composition comprising a therapeutically effective amountof a selectin in association with a polyanionic compound.
 25. Thecomposition of claim 24 wherein said polyanionic compound is a dextran,heparin or a functional derivative thereof.
 26. The pharmaceuticalcomposition of claim 24 wherein said selectin is L-selectin.
 27. Amethod for enhancing the sensitivity of an assay for the detection of aselectin in a test sample, comprising supplementing said test samplewith a polyanionic compound selected from the group consisting ofheparin, dextran, dextran sulfate, heparan sulfate, pentosan sulfate,and hyaluronate.